The hepatitis C virus (HCV) p7 protein forms an amantadine-sensitive ion channel required for viral replication in chimpanzees, though its precise role in the life cycle of HCV is unknown. In an attempt to gain some insights into p7 function, we examined the intracellular localization of p7 using epitope tags and an anti-p7 peptide antibody, antibody 1055. Immunofluorescence labeling of p7 at its C terminus revealed an endoplasmic reticulum (ER) localization independent of the presence of its signal peptide, whereas labeling the N terminus gave a mitochondrial-type distribution in brightly labeled cells. Both of these patterns could be visualized within individual cells, suggestive of separate pools of p7 where the N and C termini differed in accessibility to antibody. These patterns were disrupted by preventing signal peptide cleavage. Subcellular fractionation revealed that p7 was enriched in a heavy membrane fraction associated with mitochondria as well as normal ER-derived microsomes. The complex regulation of the intracellular distribution of p7 suggests that p7 plays multiple roles in the HCV life cycle either intracellularly or as a virion component.